CHAPTER 20 Electron Transport and Oxidative Phosphorylation

20-1 Where in the cell do electron transport and oxidative phosphorylation occur?
 Mitochondrial Functions Are Localized in Specific Compartments

1. Processes of electron transport and oxidative phosphorylation are membrane associated

2. In eukaryotic cells, electron transport and oxidative phosphorylation are localized in
mitochondria, which are also the sites of TCA cycle activity and fatty acid oxidation
3. The Mitochondrial Matrix Contains the Enzymes of the TCA Cycle
4. Electrons carried by reduced coenzymes (NADH, FADH2) are passed through a chain of
proteins and coenzymes to drive the generation of a proton gradient across the inner
mitochondrial membrane
5. Oxidative phosphorylation：the proton gradient runs downhill to drive the synthesis of ATP
6. It all happens in or at the inner mitochondrial membrane
20-2 How is the electron-transport chain organized?
 The Electron-Transport Chain Can Be Isolated in Four Complexes
1. The electron-transport chain involves several different molecular species, including：
 Flavoproteins, which contain tightly bound FMN or FAD as prosthetic groups and which
may participate in one- or two-electron transfer events
 Coenzyme Q, also called ubiquinone which can function in either one- or two-electron
transfer reactions
 Cytochromes are one-electron transfer agents in which the heme iron is converted from
Fe2+ to Fe3+ and back
 A number of iron–sulfur proteins, which participate in one-electron transfers involving
the Fe2+ to Fe3+
 Protein-bound copper, a one-electron transfer site that converts between Cu+ and Cu2+
2. flavoproteins, cytochromes, and iron–sulfur proteins possess electron-transferring
prosthetic groups
3. the membranes containing the electron-transport chain results in the isolation of four
distinct protein complexes, and the complete chain can thus be considered to be composed
of four parts: (I) NADH–coenzyme Q reductase, (II) succinate–coenzyme Q reductase, (III)
coenzyme Q–cytochrome c reductase, and (IV) cytochrome c oxidase
 4. if prokaryote, all in plasma membrane except Cytc that is membrane-bound 4 complexes
are indepentent

 Complex I Oxidizes NADH and Reduces Coenzyme Q → NADH-Coenzyme Q oxidoreductase

1. this complex (L-shaped complex) transfers a pair of electrons from NADH to coenzyme Q
2. complex I also known as NADH oxidoreductase or NADH dehydrogenase
3. The complex involves flavin mononucleotide (FMN), and Fe-S clusters
4. Pathway：NADH → FMN → Fe-S → UQ → FeS → UQ (coenzyme Q)
5. Complex I Transports Protons from the Matrix to the Cytosol：
 four H+ transported per two electrons passed from NADH to UQ
 The cytosolic side, where H+ accumulates, is referred to as the P (positive) face；the
matrix side is the N (negative) face
 Complex II Oxidizes Succinate and Reduces Coenzyme Q → Succinate-CoQ oxidoreductase
1. Complex II is perhaps better known by succinate dehydrogenase, the only TCA cycle enzyme
that is an integral membrane protein in the inner mitochondrial membrane
2. Four subunits, including 2 Fe-S proteins
 3. Pathway：succinate → FADH2 → Fe2+ → UQH2
4. Net rxn: Succinate + UQ → fumarate + UQH2
5. Proton transport does not occur in this complex (no sufficient to drive transport of protons
across inner mitochondria membrane)
6. FADH2 → 2ATP；NADH → 3ATP
7. Other flavoproteins (sn-glycerophosphate DH) can also supply electrons to UQ
8. The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves
reduction of enzyme-bound FAD

 Coenzyme Q
1. Coenzyme is a small hydrophobic molecule that is dissolved in the inner mitochondria
membrane
2. It act as an electron carrier and shuttle electrons from complex I/II to complex III
 Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c
1. CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle
2. Cytochromes were first named and classied on the absorption spectra
3. Complex III have some important structure：
 cytochrome c、cytochrome bL and b H
 Fe-S clusters
 4. Complex III Drives Proton Transport
 first half of Q cycle
A. the process by which electrons travel from the QH2 to cytochrome c is known Q
cycle
B. this cycle begins when QH2 binds to complex III. Upon binding, the two electrons
follow different pathways
C. one e- moves onto the Fe-S group and then is transferred onto the heme group of
cytochrome c. It is then diffuses away and travel to complex IV
D. the second e- moves onto the heme groups of cytochrome
E. leaves UQ – (semiquinone) in Qn site
 second half of Q cycle
A. UQH2 oxidized at the Qp site, one electron being passed to cytochrome c1 and the
other transferred to heme bL and then to heme bH
B. the bH electron is transferred to UQ - at the Qn site
C. With the addition of two H1 from the mitochondrial matrix, this produces a
molecule of UQH2, which is released from the Qn site and returns to Q pool
 Complex III takes up two protons on the matrix side of the inner membrane and releases
four protons on the cytoplasmic side for each pair of electrons that passes through the
Q cycle (Q cycle is an unbalanced proton pump)

 Cytochrome
1. This is a small soluble protein that transfer electrons between complex III and complex IV
2. Cytochrome c is the only one of the mitochondrial cytochromes that is water soluble
3. Cytochrome c binds on the intermembrane space face
4. it migrates along the membrane surface in the reduced state, carrying electrons to
cytochrome c oxidase, the fourth complex of the electron-transport chain
 Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side
1. Complex IV is called cytochrome c oxidase because it accepts e- from cytochrome c and
directs them to the four-electron reduction of O2 to form H2O
2. Bovine cytochrome c oxidase 13 subunit → 3largest subunit (I, II, III)
 Subunit I contain proton channels and redox centers
 Subunit II is common to most organism
  Subunit III carry out O2 reduction and proton transport
 I, II, III are encoded by mitochondria DNA；and others are nuclear DNA encoded
3. Electron Transfer in Complex IV Involves Two Hemes and Two Copper Sites
 pathway：binding of cytochrome c→Cyt c→CuA→heme a→CuB/heme a3→O2
 cytochrome c binds on the intermembrane space, transferring e- through 2copper and
2 heme centers to reduce oxygen on the matrix side of the membrane
 electrons from cytochrome c are used in a four e- reduction of O2 to produce 2H2O
 O2 is the terminal acceptor of electrons in ETCs

複合體（Complex）
名稱 構成 介紹
Complex I NADH-Q oxidoreductase NADH-dehydrogenase A. L-shaped
FMN B. 含最多 subunit
Fe-S centers C. 分子量最大
D. 接收 NADH 的電子起始墊子傳遞鏈
ComplexII Succinate-Q Succinate dehydrogenase A. 含最少次單元體（subunit）
oxidoreductase FAD（covalent） B. 分子量最小
Fe-S centers C. 當複合體將 Succinate 轉 Fumarate
b-type heme 時，會產生 FADH2，FADH2 再將電子
傳給 Complex II 起始傳遞鏈
ComplexIII Q-cytochrome c cytochrome bc1 complex
oxidoreductase 2 b-type hemes
Rieske Fe-S center
c-type heme（cyt c1）
ComplexIV cytochrome c oxidase cytochrome aa3 complex 會將 O2 還原成 H2O
2 a-type hemes
Cu ions
複合體總整

複合體 Complex I Complex II Complex III Complex IV

輔基 FMN FAD Heme bH、Heme bL、Heme C1 Heme a、Heme a3

複合體
Fe-S Rieske Fe-S Fe-S CuA、CuB
包含的輔基
A. bH 表「親和性高」，bL 表「親和性低」。親和性為蛋白質與蛋白質之間結合力的大小
B. Complex IV 是唯一需要銅離子（CuA、CuB）的複合體
C. Rieske Fe-S 與 Fe-S 的不同：Rieske Fe-S 的 Fe 是連接 histidine- SH groups 而非 cysteine-SH groups
Complex I NADH + Q + 5H+ → NAD + QH2 + 4H+
進行 Complex II Succinate + Q → Fumarate + QH2
反應 Complex III 2Cyt C（氧化態）+ QH2 + 2H+ → 2Cyt C（還原態）+ Q + 4H+
Complex IV 4Cyt C（還原態）+ 8H+ + O2 → 4Cyt C（氧化態）+ 4H+ + 2H2O

比較項目 Complex I Complex II Complex III Complex IV

跨膜 O X O O
質子汞 O X O O
與 Q 相鄰 O O O X
本身性質 還原酶 還原酶 還原酶 氧化酶
從 Complex I 到 Complex IV，酵素由脫氫酶轉為氧化酶，而粒線體膜電位也由負轉為正
 Electron Transfer Energy Stored in a Proton Gradient: The Mitchell Hypothesis
1. energy stored in a proton gradient across the inner mitochondrial membrane by electron
transport drives the synthesis of ATP in cells
2. Flow of protons down this electrochemical gradient, an energetically favorable process,
drives the synthesis of ATP

20-3 How does a proton gradient drive the synthesis of ATP?

 ATP Synthase Is Composed of F1 and F0
1. Proton diffusion through ATP synthase drives ATP synthesis
2. The spheres observed in electron micrographs make up the F1 unit, which catalyzes ATP
synthesis → protein subunit function with 𝛼3, 𝛽, 𝛾, 𝛿, 𝜀
 3. F1 consists of five polypeptide chains named a, b, g, d, and e, with a subunit stoichiometry
4. These F1 spheres are attached to an integral membrane protein aggregate called the F0
unit → F0 forms the transmembrane pore or channel
5. oligomycin sensitivity-conferring protein (OSCP), slender stalk that connects F0 in the
membrane with F1

 The Catalytic Sites of ATP Synthase Adopt Three Different Conformations

1. In walker’s crystal structure of F1, one of the 𝛽 subunits contains AMP-PNP, one contains
ADP, and the third site is empty → the 3 states of Boyer’s model

 Boyer’s 18O Exchange Experiment Identified the Energy-Requiring Step

1. The binding change mechanism for ATP synthesis by ATP synthase
2. This model assumes that F1 has 3 interacting and conformationally distinct active sites
3. Synthesis of ATP：
 initiated by binding of ADP and Pi to an L site
 energy-driven conformational change converts L to T & T to O and O to L
 ATP is synthesized at the T site and released from the O site
4. Two additional passes through this cycle produce two more
ATPs and return the enzyme to its original states
 Proton Flow Through F0 Drives Rotation of the Motor and Synthesis of ATP
1. Protons entering the inlet half-channel in the a subunit are transferred to binding sites on c
subunit
2. Rotation of the c-ring delivers protons to the outlet half-channel in the a-subunit
3. Flow of protons throught the structure turns the rotator and drives the cycle of
conformational changes in 𝛽 (F1) that synthesize ATP
 Racker and Stoeckenius Confirmed the Mitchell Model in a Reconstitution Experiment
1. The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by
Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis

 Inhibitors of Oxidative Phosphorylation Reveal Insights About the Mechanism

1. Rotenone inhibit complex I and helps natives of the Amazon rain forest catch fish
2. Cyanide, azide, CO inhibit complex IV, binding tightly to the ferric form (Fe3+) of a3
3. Oligomycin and DCCD are ATP synthase inhibitors
 Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthesis

1. uncouplers are hydrophobic molecules with a dissociable proton

2. across membrane, it carriers proton to dissipate H+ gradient
3. they disrupt the tight coupling between electron transport and the ATP synthase
4. not direct binding to ETC
 ATP–ADP Translocase Mediates the Movement of ATP and ADP Across the Mitochondrial
Membrane
1. ATP and ADP is do not readily cross biological membranes
2. The charge on ATP at pH 7.2 or so is about -4, and the charge on ADP at the same pH is
about -3
 3. ATP movement out is favored because the cytosol is + relative to the – matrix
4. So every ATP transported out costs one H+
5. Net movement of a negative charge out- equivalent to a H+ going in
6. Finally, membrane potential is diminished by this translocase and operate with energy cost

20-4 What is the P/O ratio for mitochondrial oxidative phosphorylation?

 P/O ratio
1. The number of ATP formed in oxidative phosphorylation per two e- flowing through a
defined segment of the ETC
2. If a transport chain yields 10H+ (4+4+2) pumped out per electron pair from NADH to oxygen
3. If 4H+ flow back into matrix per ATP to cytosol → 10/4 = 2.5 electrons entering as NADH
4. For electrons entering as succinate (FADH2), about 6H+ (4+2) pumped per electron pair to
oxygen → 6/4 = 1.5 for electrons entering as succinate
20-5 How are the electrons of cytosolic NADH Fed into electron transport?
 Introduction：
1. The NADH is produced by glycolysis and TCA cycle in cytosol
2. But most NADH used in electron transport is needed in matrix and NADH doesn’t cross the
inner mitochondria membrane
3. Shuttle systems affect e- movement without actually carrying NADH

 The Glycerophosphate Shuttle Ensures Efficient Use of Cytosolic NADH

1. 在糖解作用中 NADH 的形成步驟是 G3P → 1,3 BPG
2. In the cytoplasm, NADH in glycolysis is oxidized back into NAD+ by reducing DHAP into G3P
3. This reaction is catalyzed by glycerol-3-phosphate dehydrogenase in cytoplasm
4. Glycerophosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD
5. The G3P then moves into intermembrane space of the mitochondria, where it is oxidized
back into DHAP by glycerol-3-phosphate dehydrogenase (membrane-bound)
 6. cytosolic NADH can be used to produce mitochondrial [FADH2] and, subsequently, UQH2
7. As a result, cytosolic NADH oxidized via this shuttle route yields only 1.6 molecules of ATP
8. It is essential irreversible, even when [NADH/NAD] is low, the cycle operates effectively
9. 存在位置：腦部、骨骼肌等耗能較多的器官組織中（心肌細胞中缺乏）
 The Malate–Aspartate Shuttle Is Reversible

1. Malate－aspartate shuttle 共五種分子參與：Malate、Oxaloacetate、α-KG、Aspartate、

Glutamate
2. 草醋酸（oxaloacetate；OAA）與 NADH 皆無法穿過粒線體內膜
3. NADH+H+釋放能量，使 OAA 經 malate dehydrogenase 轉變為 malate
4. Malate 與α-ketoglutarate 透過粒線體內膜上的 malate-α-ketoglutarate antiporter 進出
粒線體基質
5. Malate 接著在基質中行逆向反應產生 OAA
6. OAA 則會與 Glutamate 作用，經轉胺作用形成α-KG 和 Aspartate，而α-KG 由 antiporter
進出 matrix
7. Aspartate 會由 Aspartate-glutamate antiporter 運出 matrix 進入 cytosol，進行轉胺作用
形成 OAA 與 glutamate，而 glutamate 則由 antiporter 進入 matrix
 The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used
1. In bacteria no mitochondria、no extra H+ used to export ATP to cytosol、not carry out
ATP/ADP exchange
2. P/O 值是估計值，回隨代謝狀態改變而變動
20-6 How do mitochondria mediate apoptosis?
 Cytochrome c Triggers Apoptosome Assembly
1. Mitochondria play a significant role in apoptosis, the programmed death of cells, partly by
regulating some of the apoptotic activator molecules, e.g. cytochrome c
2. Cytochrome c is anchored at the inner mitochondrial membrane by association with
cardiolipin
3. The peroxidase activity of cytochrome c oxidizes a cardiolipin lipid chain, releasing
cytochrome c from the membrane